Visualizing biomolecular processes has been enhanced by combining fluorophores with bioorthogonal chemistry, resulting in new tools to study the complex biochemical milieu of living cells and organisms. (N. K. Devaraj, et al., Angew. Chem. 2009, 121, 7147-7150; M. Boyce and C. R. Bertozzi, Nat. Methods 2011, 8, 638; and C. L. Droumaguet and C. Wang, Q. Wang, Chem. Soc. Rev. 2010, 39, 1233.) The resulting probes have been applied to image glycosylation and phospholipid uptake, cellular proteins, and intracellular drug distribution. (P. Shieh, et al., J. Am. Chem. Soc. 2012, 134, 17428; S. T. Laughlin, et al., Science 2008, 320, 664; M. J. Hangauer and C. R. Bertozzi, Angew. Chem. 2008, 120, 2353; J. Yang, et al, Angew. Chem. 2012, 124, 7594-7597; K. Lang, et al., Nat. Chem. 2012, 4, 298; D. S. Liu, et al., J. Am. Chem. Soc. 2012, 134, 792; N. K. Devaraj, et al., Bioconjug. Chem. 2008, 19, 2297; J. Z. Yao, et al., J. Am. Chem. Soc. 2012, 134, 3720; K. S. Yang, et al., Angew. Chem. 2012, 124, 6702-6707; G. Budin, et al., Angew. Chem. 2011, 123, 9550-9553; T. Reiner, et al, Chembiochem 2010, 11, 2374.)
Bioorthogonal “click” chemistries are widely used in chemical biology for a myriad of applications such as activity based protein profiling, crosslinking of proteins, monitoring cell proliferation, generation of novel enzyme inhibitors, monitoring the synthesis of newly formed proteins, protein target identification, and studying glycan processing. Perhaps the most fascinating applications involve using these bioorthogonal chemistries to assemble molecules in the presence of living systems such as live cells or even whole organisms (Baskin et al., 2007, Proc Natl Acad Sci USA, 104, 16793-7; Laughlin et al., 2008, Science, 320, 664-7; Prescher and Bertozzi, 2005, Nat Chem Biol, 1, 13-21; Neef and Schultz, 2009, Angew Chem Int Ed Engl, 48, 1498-500; Ning et al., 2008, Angewandte Chemie-International Edition, 47, 2253-2255). These latter applications require that the chemistry be non-toxic and possess kinetics that allow fast reaction to occur with micromolar concentrations of reagents in a time span of minutes to hours.
To fulfill these criteria, various “copper-free” click chemistries have been reported, such as the strain-promoted azide-alkyne cycloaddition and the Staudinger ligation, to react with azides on the surface of live cells both in culture and in in vivo systems such as mice and zebrafish (Prescher and Bertozzi, 2005, Nat Chem Biol, 1, 13-21). However, to date, the application of “click” chemistry in living systems, has been largely limited to extracellular targets and no technique has shown reliable ability to specifically label and image intracellular targets (Baskin and Bertozzi, 2007, QSAR Comb. Sci., 26, 1211-1219). The reasons for this are likely several. In addition to fulfilling the stability, toxicity, and chemoselectivity requirements of “click” chemistry, intracellular live cell labeling requires reagents that can easily pass through biological membranes and kinetics that enable rapid labeling even with the low concentrations of agent that make it across the cell membrane. Additionally, a practical intracellular bioorthogonal coupling scheme would need to incorporate a mechanism by which the fluorescent tag increases in fluorescence upon covalent reaction to avoid visualizing accumulated but unreacted imaging probes (i.e. background). This “turn-on” would significantly increase the signal-to-background ratio, which is particularly relevant to imaging targets inside living cells since a stringent washout of unreacted probe is not possible.
In previous years a number of elegant probes have been introduced whose fluorescence increases after azide-alkyne cycloaddition or staudinger ligation coupling reactions (Sivakumar et al., 2004, Org Lett, 6, 4603-6; Zhou and Fahmi, 2004, J Am Chem Soc, 126, 8862-3; Hangauer and Bertozzi, 2008, Angew Chem Int Ed Engl, 47, 2394-7; Lemieux et al., 2003, J Am Chem Soc, 125, 4708-9). Most of these strategies either require a reactive moiety intimately attached to the fluorophore thus requiring synthesis of new fluorophore scaffolds or take advantage of a FRET based activation requiring appendage of an additional molecule that can act as an energy transfer agent. Furthermore, most probes utilizing these popular coupling schemes have to date been unable to label intracellular targets in live cells.
The bioorthogonal Diels-Alder reaction is compatible with aqueous environments and has second order rate constants that are known to be enhanced up to several hundred-fold in aqueous media in comparison to organic solvents. (Rideout D C et al., 1980, J Am Chem Soc 102:7816-7817; Graziano G, 2004, J Phys Org Chem 17:100-101). Many Diels-Alder reactions are reversible, therefore, they may not be suitable for biological labeling. (Kwart et al., 1968, Chem Rev 68:415-447), however, the inverse electron demand Diels-Alder cycloaddition of olefins with tetrazines results in irreversible coupling giving dihydropyridazine products. During this reaction, dinitrogen is released in a retro Diels-Alder step. (Sauer J et al., 1965, Chem Ber 998:1435-1445). A variety of tetrazines and dienophiles including cyclic and linear alkenes or alkynes have been studied in this reaction. Selection of the appropriate reaction partners, allows for tuning of the coupling rate by several orders of magnitude. (Balcar J et al., 1983, Tel Lett 24:1481-1484; Thalhammer F et al., 1990, Tel Lett 47:6851-6854). See also US 2006/0269942, WO 2007/144200, and US 2008/0181847.